The neural crest provides a unique population of migratory stem cells with which to study a variety of cell and neural developmental processes. Neural crest cells emerge from the neuroepithelium in development and transform into a mesenchymal/migratory population. Several inhibitory molecules have been shown to play important roles in neural crest migration including the chemorepulsive molecules Slit 1-3. Slit molecules were initially discovered as axonal guidance molecules; however, recently they have been also shown to be true tumor suppressors and affect cell proliferation in cancer cells. Neural crest cell migration has been repeatedly likened to the process of cancer metastasis and cell invasion. Slit molecules attenuate cancer progression by regulating beta-catenin expression and are also able to negatively regulate metastasis. Results from our previous grant demonstrated that Slit chemorepellant molecules impair neural crest cell migration and alters neural crest cells cytoskeletal organization towards a non- migratory phenotype. However, we still do not know the precise role that Slit molecules play on pre-migratory neural crest cells. Do Slit molecules prevent neural crest delamination in a manner analogous to the way Slits prevent tumor metastasis? If so, what are the molecular mechanisms that allow Slit molecules to prevent the delamination of the pre-migratory neural crest cells? Do Slits modulate pre-migratory neural crest cell proliferation? The significance of this proposal is that it will determne whether or not Slits have an effect on neural crest delamination during the epithelial-to- mesenchymal transition (EMT) via their tumor suppressor mechanisms and/or if Slits modulate pre-migratory neural crest proliferation. The approach of this new proposal is to study of the role of Slit molecules in the pre-migratory neural crest cell using cell biology and genomic methods. The findings from this project will: 1) expand the current knowledge on the role of the tumor suppressor Slits in neural crest cell proliferation; 2) determine if pre-migratory neural crest cels need to silence Slits before delamination; and, 3) provide insight into tumor suppressor activity by elucidating the role of Slits in cell migration.